SARS-CoV-2 RNA-dependent RNA polymerase as a target for high-throughput drug screening

The ongoing COVID-19 pandemic caused by the SARS-CoV-2 has necessitated rapid development of drug screening tools. RNA-dependent RNA polymerase (RdRp) is a promising target due to its essential functions in replication and transcription of viral genome. To date, through minimal RNA synthesizing machinery established from cryo-electron microscopy structural data, there has been development of high-throughput screening assays for directly screening inhibitors that target the SARS-CoV-2 RdRp. Here, we analyze and present verified techniques that could be used to discover potential anti-RdRp agents or repurposing of approved drugs to target the SARS-CoV-2 RdRp. In addition, we highlight the characteristics and application value of cell-free or cell-based assays in drug discovery.


Assay
Type Target Analysis Screening result Ref.
turns of duplex RNA that engage the tripartite complex [38]. They showed a protruding RNA and long helical extensions in the nsp8 which might be sliding poles. The extensions slide along exiting RNA to block untimely dissociation of the nsp12, thus accounting for the functions played by the nsp8 in conferring processivity to the nsp12 during replication [38]. So far, there have been more than twenty structural data on the SARS-CoV-2 RdRp and associated factors [13].

Assays used to screen for inhibitors of SARS-CoV-2 RdRp
The establishment of the SARS-CoV-2 nsp12-nsp8-nsp7 complex has facilitated drug discovery in cell-free and cell-based systems. Table 1 summarizes current assays used to screen for potential inhibitors of the SARS-CoV-2/SARS-CoV RdRp. Primer extension assay Nucleoside analog inhibitors (NIs) have been in clinical treatment for over 50 years, and have become the mainstay of antiviral treatment options [51]. Since the NIs are prodrugs, they undergo cleavage in the liver via hepatic enzymes and are chemically transformed into a triphosphate form that directly acts on highly conserved and active RdRp sites [52]. Eventually, the NIs get incorporated into nascent RNA causing a lethal mutation or termination of chain elongation [53]. Currently, remdesivir (GS-5734), favipiravir, molnupiravir, ribavirin and penciclovir are promising NIs against RdRp for COVID-19 [53][54][55][56]. Hence, different studies have quantified and assessed the efficiency of incorporation of the NIs by SARS-CoV-2 RdRp. For instance, Lu et al. developed a simplified and robust in vitro nonradioactive primer extension assay to screen for NIs against RdRp ( Figure 2) [41]. Briefly, the assay employs an RNA template corresponding to the 3 end of the SARS-CoV-2 RNA genome sequence and a complementary RNA primer containing a 5 end fluorescent label to construct a primer-template (P/T) duplex [41]. To perform the primer extension reaction, the annealed P/T complex is incubated with pre-assembled nsp12-nsp8-nsp7 complex in the reaction buffer, and the reaction is initiated by addition of natural ribonucleotide triphosphates as well as tested NIs to perform the incorporation of the NIs and chain-termination assays, respectively [41]. The P/T duplex extension products are then analyzed under denaturing polyacrylamide gel electrophoresis (urea-PAGE) following termination by the quenching solution [57]. Discrimination values, defined as relative efficiency of incorporation of the NIs versus natural nucleotides by the SARS-CoV-2 RdRp, are determined by estimation of the K 1/2 values (the concentrations of substrates causing 50% primer extension), and are used to compare the chain-termination ability of a range of NIs [41,57]. The data showed higher incorporation efficiency of remdesivir triphosphate (RDV-TP) compared with that of ATP, and quadruple addition of RDV-TP gave rise to a robust termination [41]. To date, several primer extension assays using SARS-CoV-2 RdRp have been performed to examine the antiviral activity of NIs [42,43]. For instance, triphosphates of carbovir, entecavir, ganciclovir and stavudine acted as immediate chain terminators in the polymerase extension reaction [42]. Furthermore, this method was previously used to evaluate the antiviral ability of the NIs in targeting Zika and dengue virus RdRp and was applied to measure potential mitochondrial toxicity of NIs by human mitochondrial DNA-dependent RNA polymerase (POLRMT) [57][58][59]. In summary, primer extension assay has been a valuable method in screening nucleotide-based inhibitors of RdRp.

Fluorescence-based assay
To accelerate the discovery of specific SARS-CoV-2 RdRp inhibitors, there is huge potential in the use of a reliable and cost-effective high-throughput screening (HTS) assay for antiviral compounds. To evaluate inhibitors 10  of SARS-CoV RNA synthesis, a fluorescence-based HTS assay has been validated through screening of a small chemical library (1520 compounds) of US FDA approved drugs [44]. In this assay, a homopolymeric adenine RNA template and the SARS-CoV RNA polymerase complex are incubated and then mixed with prepared compounds plus UTP, which acts as a nucleotide substrate. The generated dsRNAs are then detected by addition of a fluorescent intercalant agent (Picogreen) [44,60]. Picogreen is originally developed for staining and quantifying dsDNA but can bind dsRNAs in comparison to ssRNAs. The effect of putative compounds is shown by the fluorescence readouts ( Figure 3). Besides the Picogreen dsDNA fluorescent reagent, previous studies have shown other fluorescent dyes used to discriminate dsRNAs from ssRNAs. For instance, SYTO 9 is employed to monitor real-time polymerization of the Zika virus RdRp [61], while Quantiflour dsDNA system is shown to be an ideal dye for quantification of dsRNAs formed in bacteriophage 6 RdRp reactions [45]. Recently, a reproducible and reliable HTS model has been developed to identify SARS-CoV-2 RdRp inhibitors in a 96-well plate, which combines a uracil-rich selfpriming RNA with fluorometric measurement of dsRNA. This model has demonstrated two non-nucleoside analog inhibitors (NNIs) C646 and BH3I1 block the RdRp activity with an IC 50 value of 14.31 μM and 56.09 μM respectively and has been applied to screen for potential RdRp inhibitors from a custom synthetic chemical and natural product library, offering an efficient platform for identifying and confirming novel molecules targeting the SARS-CoV-2 RdRp, especially NNIs [46].

Strand displacement assay
Bertolin et al. designed a fluorescence resonance energy transfer-based strand displacement assay amenable to HTS for discovering small molecules targeting SARS-CoV-2 RdRp in a 384-well plate [47]. The activity of strand displacement for DNA/RNA polymerases refers to the ability to displace the pre-existing DNA/RNA fragment downstream while synthesizing the new fragment [62,63]. Utilizing this activity of RdRp, the RNA substrate is formed which consists of a RNA template labeled with a Cy3 fluorophore on its 5 end, an annealed primer, and an annealed quencher strand close to the fluorophore. After incubation of RdRp with compounds, the polymerization reactions are started by the addition of substrate mix. When RdRp extends the primer, it can drive strand displacement of the quencher strand downstream leading to growing fluorescent signals ( Figure 4). The authors have identified three novel NNIs against RdRp (C646, BH3I-1 and GSK-650394) and demonstrated the anti-RdRp efficacy of suramin and suramin-like molecules [47]. On the other hand, it is noteworthy that the above cell-free assays employ nsp12 along with its cofactors, nsp7 and nsp8. The cofactors could act as a mixture by simply mixing an optimized ratio of purified nsp7 and nsp8 [41,42,64]   or as a fusion protein by introducing a 6 × histidine or GSGSGS linker between the two subunits [36,44], thus supporting the constitution of the minimal RNA polymerase complex.

Cell-based reporter assay
The cell-free assays have presented several insuperable obstacles. First, the assays require conversion of the NIs prodrugs into 5 -triphostrates, which is a normal metabolic process in cells [65]. However, in vitro synthesis of active triphosphate forms is a complicated and time-consuming chemical process, which impedes the development of the cell-free assays for drug screening [65]. Besides, it is difficult to assess cellular permeability of examined compounds without a cell system. In addition, purified recombinant enzymes do not always undergo proper folding and thus might have incorrect structural conformation [66]. To circumvent the challenges associated with the cell-free assays, a cell-based reporter assay targeting SARS-CoV-2 RdRp has been proposed on the basis of previous effective systems [67,68]. The reporter system is divided into two components: a Gaussia-luciferase (Gluc) reporter plasmid and plasmids bearing RNA synthesis genes made up of nsp7, nsp8 and nsp12 [48]. The Gluc expression reporter plasmid has the Gluc gene flanked by 5 and 3 untranslated regions of the SARS-CoV-2, which produces positive-strand of vRNA encoding the Gaussia luciferase [65]. Transcription of the Gluc gene is modulated by CMV promoter [65]. Within the HEK293T cells that are transfected with appropriate ratios of CoV-Gluc, nsp12, nsp7 and nsp8 plasmids, the untranslated regions of the Gluc mRNA are recognized by the SARS-CoV-2 RdRp and then the mRNA is amplified by the viral RdRp. The amplification occurs through the minus-strand stage, initiating extensive elevation of the Gluc mRNA and protein ( Figure 5) [48,49]. After incubation with selected compounds, cell supernatant is collected and used to measure the Gluc activity, as the relative activity of the SARS-CoV-2 RdRp is proportional to the intensity of the luciferase activity [49]. Statistical analyses show that the Z' factor, a frequently used parameter for validating the sensitivity and accuracy of HTS assays [69], meets the threshold required for the techniques [65]. Taken together, the cell-based assay with Gluc as the reporter gene is a feasible platform for testing viral polymerase inhibitors in HTS systems. This is achieved by simply detecting the luciferase signals.

Conclusion
So far, the urgent need to contain the SARS-CoV-2 pandemic has propelled the rapid advances in drug development strategies. RdRp is deemed as the Achilles' heel in the SARS-CoV-2 due to its essential functions in viral lifecycle.
Owing to high-resolution cryo-EM structural analysis, minimal RNA synthesizing machinery has been elucidated as a complex between nsp7 with nsp8 and nsp12. Drug discovery assays targeting this tripartite complex have been developed at an unprecedented rate. Undeniably, a variety of computational screening approaches including ligand-based and structure-based drug screening have become a promising alternative for drug discovery processes to speed up the development of candidates against RdRp [70]. Several reviews have presented in silico studies on identifying potential RdRp inhibitors [71][72][73]. However, these compounds determined by in silico strategies still require further experimental evaluations to test their antiviral potency. For in vitro assays, traditional methods employ 32 P-labeled nucleotides or primers that integrate into nascent RNA strands to analyze the enzymatic characteristics of the viral RdRp [45]. Although highly sensitive, these radioactive methods are limited because of the health concerns and environmental safety. The limitations hinder longer-term development and broader application of these methods. In contrast, the primer extension assay, fluorescence-based assay and strand displacement assay present safer alternatives since they do not utilize radioactive substances. The assays use fluorescently labeled RNA substrates and the fluorescent dye, which do not require specialized equipment and handling procedures. Non radioactivity is a common attribute associated with the above cell-free assays and they are beneficial in diverse ways. First, as a popular biochemical method to characterize the activity of the viral polymerases, primer extension assay provides a molecular basis to better comprehend the mechanism of action of promising antiviral compounds that target the SARS-CoV-2 RdRp. For instance, Sofosbuvir inhibits the synthesis of SARS-CoV-2 RNA as a chain terminator by polymerase extension reaction [74]. In addition, Gordon et al. observed that incorporation of RDV-TP at position i causes termination of RNA synthesis at position i+3, which is regarded as delayed chain-termination pattern [75]. Subsequent studies confirmed similar behavior of RDV-TP, and pointed that the RDV-TP-specific delayed chain-termination pattern varies with different template sequences [41,76]. On the other hand, miniaturization and automation in the fluorescence-based assays enhance the discovery and identification of SARS-CoV-2 RdRp inhibitors from small-molecule compound libraries. It is feasible to anticipate increased output by scaling up the formats to 384 and 1536-well plates. Along with the benefits of the small volume of the reaction mixture and minimal operation, the assay utilizes economical homopolymeric RNA as a template instead of labeled synthetic heteropolymeric RNA [61]. Additionally, strand displacement assay enables real-time detection of RdRp activity avoiding the limitation of depending only on end point values [47].
In contrast to cell-free assays, a typical cell environment is the most significant advantage associated with the cell-based reporter assay. Instead of preparing the triphosphate forms of the NIs, the cell-based reporter assay is capable of directly testing nucleotide prodrugs. In addition, it is possible to simultaneously evaluate cellular permeability and cytotoxicity of the drugs. The assay also mimics a physiological environment to form the RNA synthesis complex, evading the often tedious in vitro recombinant protein purification protocols. To improve the consistency and convenience of the experimental design at the level of transient transfection, the next process is to establish stable cell lines that could co-express all of the integral components involved in the replication and transcription of the SARS-CoV-2 RNA genome [65].
Collectively, we summarize cell-free and cell-based assays for detecting the enzymatic activity of SARS-CoV-2 nsp12-nsp8-nsp7 complex and evaluating the inhibitory effect of prospective anti-RdRp compounds. The emergence of developed and proven techniques with the target of SARS-CoV-2 RdRp has breathed new life into the course of antiviral candidate exploring, though deeper investigation is awaited. There is no dispute that HTS assays lie at the heart of the screening from the reservoir of FDA approved drugs for treatment of other diseases, a series of novel synthesized inhibitors and the molecules which have been shown potential antiviral performance in computational analyses. Furthermore, structural and functional insights into coronavirus replication and transcription will contribute to the improvement and optimization of in vitro screening schemes which can be formatted into HTS conveniently.

Future perspective
Recent studies have highlighted that the nsp14-nsp10 complex, responsible for RNA proofreading which is requisite for attainment and maintenance of large nidovirus genomes [77], should be incorporated into the screening assays that focus on the RdRp [49,65]. In complex with its activator nsp10, nsp14 has been shown to increase the fidelity of viral replication through its exoribonuclease domain, posing a relatively high resistance threshold of the RdRp to NA compounds [78,79]. By simultaneously introducing the nsp14-nsp10 and RdRp into the cell-based reporter assay, it was shown that the exoribonuclease complex, nsp14-nsp10, intensifies the resistance property of SARS-CoV-2 RdRp to NA inhibitors [65]. Therefore, considering enhanced tolerance of RdRp conferred by the proofreading capacity of exoribonuclease in seeking anti-RdRp compounds, it is necessary to expand the minimal RNA polymerase complex (nsp12-nsp8-nsp7 complex) to the nsp12-nsp8-nsp7-nsp14-nsp10 super complex.
In addition to testing the efficacy of a single candidate, these reviewed approaches could be applied to evaluate the additive or synergistic interactions between two or more potent RdRp inhibitors with different modes of action to suppress potential drug resistance and enhance antiviral potency. For instance, a combination of a NNI, corilagin (RAI-S-37) and a NI, remdesivir, exhibits additive anti-RdRp activity in the cell-based reporter assay [49]. Therefore, combined drug testing for SARS-CoV-2 RdRp might be a novel strategy for development of efficient inhibitor screening assays.
Author contributions J Wu, Z Chen, X Han, Q Chen, Y Wang and T Feng all contributed to review design, drafting the article, critical revision of content and final approval for publication. All authors have read and agreed to the published version of the article.

Acknowledgments
The authors apologize that there may be other excellent studies not included. The authors thank the reviewers for their critical review of the manuscript. and sincerely thank the Home for Researchers editorial team for providing their language editing services. Review with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Financial & competing interests disclosure
No writing assistance was utilized in the production of this manuscript.

Executive summary
• The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is acknowledged as a valuable target for drug development.

The functions of SARS-CoV-2 RdRp
• RdRp plays an indispensable role in replication and transcription of the SARS-CoV-2 genome.
• RdRp has two functional domains: a polymerase domain and a nidovirus N-terminal RdRp-associated nucleotidyltransferase (NiRAN) domain. The RdRp is dependent on nsp7 & nsp8 in RNA synthesis • Together with cofactors, nsp7 and nsp8, RdRp also referred to as nsp12 forms the basic RNA synthesizing machinery, the nsp12-nsp8-nsp7 complex. • The cryo-electron microscopy structures give more insights into the RNA synthesizing complex.

Assays used to screen for inhibitors of SARS-CoV-2 RdRp
• Primer extension assay is mainly used for discovering nucleotide-based inhibitors of RdRp.
• Fluorescence-based assay features a fluorescent intercalant agent which is capable of quantifying dsRNA.
• Cell-based reporter assay characterized by a cell environment circumvents several difficulties associated with the cell-free assays. • Strand displacement assay is a fluorescence resonance energy transfer-based strand displacement assay amenable to high-throughput screening for discovering small molecules targeting SARS-CoV-2 RdRp in a 384-well plate.

Conclusion
• It is necessary to develop high-throughput screening assays for evaluating the efficiency of RdRp inhibitors. Future perspective • The nsp14-nsp10 complex should be considered into the screening assays with the target of RdRp.
• It can be applied to test combined inhibitory performance utilizing two or more potent RdRp inhibitors with these verified techniques.